A Two-Tube Combined TaqMan/SYBR Green Assay to Identify Mycobacteria and Detect Single Global Lineage-Defining Polymorphisms in Mycobacterium tuberculosis

Abstract

When a possible case of mycobacterial infection is detected, both the clinical and public health management depend critically on whether the causative agent is Mycobacterium tuberculosis or a nontuberculous mycobacterium (NTM). In the former case, treatment and prevention of further person-to-person transmission are urgent concerns while, in the latter, infections are predominantly sporadic, derive from environmental sources of infection, and often reflect pre-existing conditions in the patient. 1-3Although there are several promising direct molecular assays for M. tuberculosis, 4 -9 culture is the most sensitive means to detect specific mycobacterial infections and remains the mainstay of diagnostic services in well-resourced laboratories. However, when cultures are detected positive and the presence of mycobacteria confirmed by acid fast staining, it is not known whether the isolated mycobacteria belong to the Mycobacterium tuberculosis complex (MTBC) or the NTM group. Furthermore, when the possibility of recent transmission of tuberculosis (TB) arises, no information is available regarding the relatedness of the isolate to other contemporary isolates with potential epidemiological connections to the new case.Cultures positive for mycobacteria are referred for further testing to address these points and, in the UK, this generally involves transmission to a reference laboratory where the caseload enables economies of scale in deploying established molecular identification and MTBC genotyping procedures. Thus, definitive recognition of

Characterization of MicroRNA Expression Profiles and Identification of Potential Biomarkers in Leprosy

“…Currently, the gold standard for leprosy diagnosis is based on clinical examination and skin biopsy. Techniques based on PCR technology and serological analysis have been developed but were not able to diagnose leprosy with acceptable sensitivity and specificity taking into consideration the different clinical forms and/or bacterial burden (11,(14)(15)(16)(18)(19)(20)(21). Accordingly, identification of biomarkers that allow the diagnosis of leprosy with a greater sensitivity and specificity is still needed.…”

“…Molecular techniques using PCR technology and serological tests were developed; however, specificity and sensitivity were limited (11,(14)(15)(16)(17)(18)21). In this context, the identification of biomarkers that allow early diagnosis of leprosy and differentiation between forms of the disease with an adequate sensitivity and specificity is still required.…”

Resuscitation-promoting Factors Reveal an Occult Population of Tubercle Bacilli in Sputum

“…stained samples and, after informed consent had been obtained, agreed to provide sputum samples for characterization of mycobacterial populations before and after onset of chemotherapy. All patients were microbiologically confirmed to have tuberculosis; in many cases early confirmation was obtained by application of an in-house real-time mycobacterial polymerase chain reaction (20).…”

[Letter to the editor] Combined FAM-Labeled Taqman Probe Detection and SYBR Green I Melting Curve Analysis in Multiprobe qPCR Genotyping Assays

“…So far, only two reports applied both types of detection in a single assay (10,11). Lind et al described the combination of the nonspecific dsDNA binding dye BOXTO with sequence-specific FAM-labeled TaqMan and LNA probes (10), and Cheah et al described a two-tube combined TaqMan/SYBR green assay with a ROX- and a Cy5-labeled probe (11).…”

“…Lind et al described the combination of the nonspecific dsDNA binding dye BOXTO with sequence-specific FAM-labeled TaqMan and LNA probes (10), and Cheah et al described a two-tube combined TaqMan/SYBR green assay with a ROX- and a Cy5-labeled probe (11). For those applications, they needed to combine specific sequence detection by fluorescently labeled probes with the generation of the total PCR amplicon readout by binding dyes.…”